tRNAVal allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei.

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick basepairs. In bacterial tRNA, 5-hydroxyuridine (ho5U) derivatives xo5U [x: methyl (mo5U), carboxymethyl (cmo5U), and methoxycarbonylmethyl (mcmo5U)] present at the wobble positions of tRNAs are responsible for recognition of NYN codon families. These modifications of U34 allow basepairing not only with A and G but also with U and in some cases C. mo5U was originally found in Gram-positive bacteria, and cmo5U and mcmo5U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei, Gram-positive bacteria and lactic acid bacteria, lacks the modification enzyme genes for xo5U biosynthesis. Nevertheless, L. casei has only one type of tRNAVal with the anticodon UAC [tRNAVal(UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNAUnd) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNAVal is unmodified and that there is no tRNAUnd expression in the cells. In addition, in vitro transcribed tRNAUnd was not aminoacylated by L. casei valyl-tRNA synthetase suggesting that tRNAUnd is not able to accept valine, even if expressed in cells. Correspondingly, native tRNAVal(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNAVal decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts.

Recombinant expression and purification of phenylalanyl-tRNA synthetase from wheat: a long-lasting poly(U)-dependent poly(Phe) synthesis system.

Synthetic genes for the two subunits of phenylalanyl-tRNA synthetase (PheRS) from wheat were expressed in Escherichia coli. When each gene was induced individually, the α subunit with a cleavable 6 × His tag at the amino terminus was largely soluble, while the ß subunit was almost completely insoluble. When the two subunits were co-expressed, a soluble fraction containing the two subunits were obtained. This was purified by a standard method in which the tag was cleaved off with a specific protease after affinity purification. As the sample contained slightly more PheRSα than PheRSß, we further resolved the sample by gel filtration to obtain the fraction that showed the size of the conventional α2ß2 tetrameric complex and contains an almost equal amount of the two subunits. The final yield was 0.6 mg per 1 liter of the culture medium, and the specific activity was 28 nmol min-1 mg-1, which was higher than that of a fraction purified from wheat germ. This recombinant PheRS was used, along with purified samples of the elongation factors and the ribosomes from wheat germ, for a poly(U)-dependent poly(Phe) synthesis reaction. The reaction was dependent on the added components and lasted for more than several hours.

Spin and charge interactions between nanographene host and ferrocene.

Ferrocene (FeCp2) was introduced as a non-magnetic guest molecule to activated carbon fibers (ACFs) as a nanographene-based host having localized spins originating from zigzag edges of graphene. The introduction of the guest molecule was confirmed by FTIR for ACFs-FeCp2 introduced at 55 (150) °C (FeCp2-ACFs-55(150)). The appearance of satellite Fe2p peaks and the increase in shake-up peak intensity of the C1s in the XPS spectrum proved the emergence of charge-transfer host-guest interaction in FeCp2-ACFs-150, supported by the red-shift of the G-band in the Raman spectrum. The six-times enhancement in the spin concentration in FeCp2-ACFs-150 compared with ACFs indicates the spin magnetism of the non-magnetic guest FeCp2+ molecule induced by a charge-transfer host-guest interaction in the nanographene host. The larger ESR linewidth than that expected from the dipolar interaction estimated by the localized spin concentration suggests the exchange interaction between the nanographene and FeCp2 spins. The narrowing of the ESR linewidth of FeCp2-ACFs-55 upon higher excitation microwave power suggests the inhomogeneity of the environment for FeCp2+ molecules in the nanographene host. The observed induction of spin magnetism by the interfacial interactions between the nanographene host and the guest molecules will be a promising strategy for developing a new class of molecular magnets.

Correlation between charge density wave phase transition and hydrogen adsorption in 1T-TaS2thin film devices.

Thin films of tantalum disulfide in the 1T-polytype structural phase (1T-TaS2), a type of metallic two-dimensional (2D) transition metal dichalcogenides (TMDs), are reactive to H2. Interestingly, in the incommensurate charge-density wave (ICCDW) phase with a metallic state, the electrical resistance of the 1T-TaS2thin film decreases when H2is adsorbed on it and returns to its initial value upon desorption. In contrast, the electrical resistance of the film in the nearly commensurate CDW (NCCDW) phase, which has a subtle band overlap or a small bandgap, does not change upon H2adsorption/desorption. This difference in H2reactivity is a result of differences in the electronic structure of the two 1T-TaS2phases, namely, the ICCDW and NCCDW phases. Compared to other semiconductor 2D-TMDs such as MoS2and WS2, the metallic TaS2has been theoretically proven to capture gas molecules more easily because Ta has a stronger positive charge than Mo or W. Our experimental results provide evidence of this. Notably, this study is the first example of H2sensing using 1T-TaS2thin films and demonstrates the possibility of controlling the reactivity of the sensors to the gas by changing the electronic structure via CDW phase transitions.

Intron-Dependent or Independent Pseudouridylation of Precursor tRNA Containing Atypical Introns in Cyanidioschyzon merolae.

Eukaryotic precursor tRNAs (pre-tRNAs) often have an intron between positions 37 and 38 of the anticodon loop. However, atypical introns are found in some eukaryotes and archaea. In an early-diverged red alga Cyanidioschyzon merolae, the tRNAIle(UAU) gene contains three intron coding regions, located in the D-, anticodon, and T-arms. In this study, we focused on the relationship between the intron removal and formation of pseudouridine (Ψ), one of the most universally modified nucleosides. It had been reported that yeast Pus1 is a multiple-site-specific enzyme that synthesizes Ψ34 and Ψ36 in tRNAIle(UAU) in an intron-dependent manner. Unexpectedly, our biochemical experiments showed that the C. merolae ortholog of Pus1 pseudouridylated an intronless tRNAIle(UAU) and that the modification position was determined to be 55 which is the target of Pus4 but not Pus1 in yeast. Furthermore, unlike yeast Pus1, cmPus1 mediates Ψ modification at positions 34, 36, and/or 55 only in some specific intron-containing pre-tRNAIle(UAU) variants. cmPus4 was confirmed to be a single-site-specific enzyme that only converts U55 to Ψ, in a similar manner to yeast Pus4. cmPus4 did not catalyze the pseudouridine formation in pre-tRNAs containing an intron in the T-arm.

Anomalous spin relaxation in graphene nanostructures on the high temperature annealed surface of hydrogenated diamond nanoparticles.

The electronic and magnetic structures of diamond nanoparticles with a hydrogenated surface are investigated as a function of annealing temperature under vacuum annealing up to 800-1000 °C. Near edge X-ray absorption fine structure (NEXAFS) spectra together with elemental analysis show successive creation of defect-induced nonbonding surface states at the expense of surface-hydrogen atoms as the annealing temperature is increased above 800 °C. Magnetization and ESR spectra confirm the increase in the concentration of localized spins assigned to the nonbonding surface states upon the increase of the annealing temperature. Around 800 °C, surface defects collectively created upon the annealing result in the formation of graphene nano-islands which possess magnetic nonbonding edge states of π-electron origin. Interestingly, extremely slow spin relaxation is observed in the magnetization of the edge state spins at low temperatures. The relaxation time is well explained in terms of a lognormal distribution of magnetic anisotropy energies instead of the classical Néel relaxation mechanism with a unique magnetic anisotropy energy, in addition to the contribution of the quantum mechanical tunnelling mechanism. The spin-orbit interaction enhanced by the electrostatic potential gradient created at the interface between the core diamond particle and surface graphene nano-islands is responsible for the slow spin relaxation.

Translational resistivity/conductivity of coding sequences during exponential growth of Escherichia coli.

Codon adaptation index (CAI) has been widely used for prediction of expression of recombinant genes in Escherichia coli and other organisms. However, CAI has no mechanistic basis that rationalizes its application to estimation of translational efficiency. Here, I propose a model based on which we could consider how codon usage is related to the level of expression during exponential growth of bacteria. In this model, translation of a gene is considered as an analog of electric current, and an analog of electric resistance corresponding to each gene is considered. "Translational resistance" is dependent on the steady-state concentration and the sequence of the mRNA species, and "translational resistivity" is dependent only on the mRNA sequence. The latter is the sum of two parts: one is the resistivity for the elongation reaction (coding sequence resistivity), and the other comes from all of the other steps of the decoding reaction. This electric circuit model clearly shows that some conditions should be met for codon composition of a coding sequence to correlate well with its expression level. On the other hand, I calculated relative frequency of each of the 61 sense codon triplets translated during exponential growth of E. coli from a proteomic dataset covering over 2600 proteins. A tentative method for estimating relative coding sequence resistivity based on the data is presented.

SfiNX: a method for assembly of protein coding sequences with high success rates.

OBJECTIVE: Concatenation of two NdeI-XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli. RESULTS: A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid. CONCLUSION: This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.

Polaron coupling in graphene field effect transistors on patterned self-assembled monolayer.

We investigated the device characteristics of a graphene field effect transistor (FET) of which interfaces were controlled by a self-assembled monolayer (SAM). Electrical transport measurements together with Raman spectroscopy characterizations for bilayer graphene (BLG) and single layer graphene (SLG) on micro-patterned SAM (mp-SAM), respectively, elucidate spatial carrier modulations on the graphene sheets driven by mp-SAM. The SLG-mp-SAM-FET device exhibits unconventional graphene p-n junction characteristics depending on the polarity of source-drain voltage. The observed characteristics can be interpreted as a velocity saturation of hole carriers coupled with polaron states, of which phonon energy is around 30 meV, on the SAM molecules at the graphene p-n junction. The SAM-based micro fabrication techniques presented in this report not only provide a spatial control of electronic properties for graphene but also lend a new perspective in the understanding of graphene-substrate interface based molecular self-assembled systems.

Preferential oxidation-induced etching of zigzag edges in nanographene.

We investigated the thermal oxidation process of nanographene using activated carbon fibers (ACFs) by thermogravimetry (TG), X-ray photoemission spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), and electrical conductance measurements. The oxidation process started from the edge of nanographene with the formation of phenol (-OH) or ether (C-O-C) groups attached to edge carbon atoms, as verified by the XPS and NEXAFS results. While the TG results indicated a decrease in the size of the nanographene sheet during the oxidation process, the intensity of the edge-state peak, i.e., the signature of the zigzag edge, decreased in the C K-edge NEXAFS spectra. This suggests that the zigzag edge preferentially reacted with oxygen and that the nanographene terminated with the thermodynamically unstable zigzag edges converted to one terminated with stable armchair edges. As the oxidation temperature increased, the activation energy for the electron hopping transport governed by the Coulomb gap variable range hopping between the nanographene sheets increased, and the tunneling barrier decreased. This change can be understood on the basis of the decrease in the size of the nanographene sheets together with the preferential etching of nanographene edges and the decrease in the inter-nanographene-sheet distance.

Heat treatment effect on the electronic and magnetic structures of nanographene sheets investigated through electron spectroscopy and conductance measurements.

The heat treatment effect on the electronic and magnetic structures of a disordered network of nanographene sheets has been investigated by in situ measurements of X-ray photoemission spectroscopy, near-edge X-ray absorption fine structure (NEXAFS), and electrical conductance, together with temperature-programmed desorption measurements. Oxygen-containing functional groups bonded to nanographene edges in the pristine sample are almost completely decomposed under heat treatment up to 1300-1500 K, resulting in the formation of edges primarily terminated by hydrogen. The removal of the oxygen-containing groups enhances the conductance owing to the decrease in the electron transport barriers between nanographene sheets. Heat treatment above 1500 K removes also the hydrogen atoms from the edges, promoting the successive fusion of nanographene sheets at the expense of edges. The decrease in the π* peak width in NEXAFS indicates the progress of the fusion reaction, that is, the extension of the π-conjugation, which agrees with the increase in the orbital susceptibility previously reported. The fusion leads to the formation of local π/sp(2) bridges between nanographene sheets and brings about an insulator-to-metal transition at 1500-1600 K, at which the bridge network becomes infinite. As for the magnetism, the intensity of the edge state peak in NEXAFS, which corresponds to the number of the spin-polarized edge states, decreases above 1500 K, though the effective edge-state spin density per edge state starts decreasing at approximately 200 K lower than the temperature of the edge state peak change. This disagreement indicates the development of antiferromagnetic short range ordering as a precursor of a spin glass state near the insulator-metal transition, at which the random network of inter-nanographene-sheet exchange interactions strengthened with the formation of the π/sp(2) bridges becomes infinite.

Anomalous metallic-like transport of Co-Pd ferromagnetic nanoparticles cross-linked with π-conjugated molecules having a rotational degree of freedom.

We investigated the electron transport in Co-Pd ferromagnetic nanoparticles (Co 16%) cross-linked with oligo(phenyleneethynylene)diethanethiolate, which consists of three rotary phenylene moieties bridged by two acetylene groups, or icosane-1,20-dithiol, which consists of one alkane chain. Although the nanoparticles cross-linked with the alkane dithiols (the latter) have extremely high electrical resistance in electron transport, the resistance of the nanoparticles cross-linked with the conjugated molecules (the former) demonstrates a linear temperature dependence from room temperature to ca. 20 K; below that temperature, it has a weak temperature-dependent residual contribution with a resistance minimum around 7 K. Computational simulations suggest that the apparent metallic-like temperature dependence at high temperatures can be explained in terms of the rotational degree of freedom of the linker molecule. The rotational motion of the constituent phenylene groups, which hinders π-conjugation along the linker molecule, becomes less excited as the temperature is lowered. The successive development of a ballistic transport path through the π-conjugated linker molecule with decreasing temperature yields the metallic-like temperature dependence observed for the bridged nanoparticles. The low-temperature resistance behaviour with a minimum is a consequence of carrier scattering by the localized Co spins of Co-Pd nanoparticles randomly ordered in a ferromagnetic state that develops below the temperature of the resistance minimum.

Magnetic edge-states in nanographene, HNO3-doped nanographene and its residue compounds of nanographene-based nanoporous carbon.

We investigated the magnetic and electronic properties of nanographene and its charge transfer effect, using near edge X-ray absorption fine structure (NEXAFS), magnetic susceptibility and ESR measurements, and elemental analysis, with the employment of nanoporous carbon, which consists of a three dimensional disordered network of loosely stacked nanographene sheets, in relation to the host-guest interaction with HNO3 as the electron-accepting guest. The adsorption of electron acceptor HNO3 decreases the intensity of the edge state peak in NEXAFS as a result of the charge-transfer-induced Fermi energy downshift, in agreement with the decrease in the edge-state spin concentration, and it also induces the structural expansion, which makes the inter-nanographene sheet distance elongated, resulting in weakening of the inter-nanographene-sheet antiferromagnetic interaction as evidenced by the decrease in the Weiss temperature. In addition, the decomposition of HNO3, which takes place with the electron-rich edge state as an oxidation catalyst, results in the creation of oxygen/nitrogen-containing functional groups bonded to the periphery of the nanographene sheets. Heat-treatment of the HNO3-ACFs under evacuation desorbs the HNO3 molecules completely, though a part of the oxygen/nitrogen-containing species remains strongly bonded to the edge even at a high temperature of ∼800 °C, according to NEXAFS and elemental analysis results. These remaining species participate in the charge transfer, modifying the electronic structure as observed with the decrease in the orbital susceptibility and the strengthening of the inter-nanographene-sheet antiferromagnetic interaction.

Isolated Spin-7/2 Species of Gadolinium (III) Chelate Complexes on the Surface of 5-nm Diamond Particles.

The magnetic characteristics of a system of triply charged gadolinium ions Gd3+ chelated with carboxyls on the surface of detonation nanodiamond (DND) particles have been studied. Gd3+ ions demonstrate almost perfect spin (S = 7/2) paramagnetism with negligible antiferromagnetic interaction between spins (Weiss temperature about -0.35 K) for a wide range of concentrations up to ~18 ions per 5 nm particle. The study of the concentration dependence of the electron paramagnetic resonance signal for DND intrinsic defects with spin ½ (g = 2.0027) shows that Gd3+ ions are located on average at a distance of no more than 1.4 nm from shallow subsurface defects with spin 1/2. At the same time, they are located (according to density functional theory calculations) at a distance of about or at least 0.28 nm from the particle surface. Magnetic studies also confirm the isolated nature of the gadolinium chelate complexes on the surface of DND particles. DND particles turn out to be an optimal carrier for high-spin 4f- ions (gadolinium) in a highly concentrated isolated state. This property makes DND-Gd particles a candidate for the role of a contrast agent for magnetic resonance imaging.

Mechanism of tRNA recognition by heterotetrameric glycyl-tRNA synthetase from lactic acid bacteria.

Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2ß2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2ß2 GlyRS, consisting of the full-length α and ß subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2ß2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2ß2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and ß subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and ß subunits of LpGlyRS together interact with the 3'-end and the acceptor region of tRNAGly, and the C-terminal domain of the ß subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.

Carrier control of graphene driven by the proximity effect of functionalized self-assembled monolayers.

We demonstrated the carrier control of graphene by employing the electrostatic potential produced by several types of self-assembled monolayer (SAM) formed on SiO(2) substrates. For single layer graphene on perfluoroalkylsilane-SAM, the stiffening of the Raman G-band indicates a large down shift of the Fermi level (∼-0.8 eV) by accumulated hole carriers. Meanwhile, aminoarylsilane-SAM accumulated electron carriers, which compensate the hole carriers doped by adsorbed molecules under the ambient atmosphere, in graphene. The present results and their theoretical analysis reveal that the use of the dipole moments of SAM molecules can systematically modulate the electrostatic potential affecting graphene without destroying its intrinsic electronic structure and let us know that the proximity effect of the SAMs is a promising way in developing graphene-based solid state electronics.

Electrically controlled adsorption of oxygen in bilayer graphene devices.

We investigate the chemisorptions of oxygen molecules on bilayer graphene (BLG) and its electrically modified charge-doping effect using conductivity measurement of the field effect transistor channeled with BLG. We demonstrate that the change of the Fermi level by manipulating the gate electric field significantly affects not only the rate of molecular adsorption but also the carrier-scattering strength of adsorbed molecules. Exploration of the charge transfer kinetics reveals the electrochemical nature of the oxygen adsorption on BLG.

The uridine to pseudouridine modification at the wobble position of eukaryotic isoleucine tRNA species is unlikely to induce mistranslation.

Replacement of a U in an RNA duplex with a pseudouridine (Ψ), in general, stabilize the duplex because of the stronger stacking interaction, even concerning the wobble pair with G. The tRNA species specific to the AUA isoleucine codon in many eukaryotes have a Ψ at the first position of the anticodon. This tRNAIle would cause mistranslation if it could recognize the AUG codon through formation of a Ψ-G base pair. Here, I propose rationales for the minimal promotive effect of the U to Ψ modification on the mistranslation of the AUG codon.

Recognition of tRNAIle with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

Magnetic sponge prepared with an alkanedithiol-bridged network of nanomagnets.

The magnetic dipole-dipole interaction between nanomagnets having huge magnetic moments can have a strength comparable to that of the van der Waals interaction between them, and it can be manipulated by applying an external magnetic field of conventional strength. Therefore, the cooperation between the dipole-dipole interaction and the applied magnetic field allows the magnetic moments of nanomagnets to be aligned and organized in an ordered manner. In this work, a network of magnetic nanoparticles connected with flexible long-alkyl-chain linkers was designed to develop a "magnetic sponge" capable of absorbing and desorbing guest molecules with changes in the applied magnetic field. The magnetization of the sponge with long-alkyl-chain bridges (30 C atoms) exhibited a 500% increase after cooling in the presence of an applied field of 7 T relative to that in the absence of a magnetic field. Cooling in a magnetic field leads to anisotropic stretching in the sponge due to reorganization of the nanomagnets along the applied field, in contrast to the isotropic organization under zero-field conditions. Such magnetic-responsive organization and reorganization of the magnetic particle network significantly influences the gas absorption capacity of the nanopores inside the material. The absorption and desorption of guests in an applied magnetic field at low temperature can be regarded as a fascinating "breathing feature" of our magnetic sponge.